Screening culture medium and screening method

ABSTRACT

According to an embodiment, there are provided a screening culture medium that enables a convenient and quick examination for the presence of Candida auris, and a screening method. According to at least one embodiment, the screening culture medium contains an enzyme substrate, in which the screening culture medium is used for screening Candida auris based on an ability to degrade the enzyme substrate, the enzyme substrate including raffinose and xylose. According to another embodiment, the screening method uses the screening culture medium.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority toPCT/JP2021/003524, filed on Feb. 1, 2021, entitled (translation),“SCREENING CULTURE MEDIUM AND SCREENING METHOD,” which claims thebenefit of and priority to Japanese Patent Application No. 2020-17999,filed on Feb. 5, 2020, which is hereby incorporated by reference in itsentirety into this application.

BACKGROUND Field

Embodiments relate to a screening culture medium and a screening methodwhich enable the identification and screening of Candida auris.

Background Art

There are more than 400 species of Candida bacteria, known as thecausative bacteria of candidiasis, of which 20 of them are clinicallyimportant. Many of them have similar morphological and biochemicalproperties and are generally difficult to be discriminated.

Studies have been carried out on a method of identifying species withrespect to the existing species of Candida bacteria. For example, PatentDocument 1 discloses a culture medium for simple identification ofCandida, which is obtained by adding 2,3,5-triphenyltetrazolium chloride(TTC) and yeast extract to a dye-added Sabouraud's culture medium. Thisculture medium for simple identification of Candida is intended to be aculture medium for carrying out discrimination between two species ofCandida bacteria, Candida albicans and Candida glabrata, according tothe colony color tone by using the difference in redox ability of thesebacterial species on the culture medium.

Patent Document 2 discloses a method of detecting the presence of aspecific microorganism bacterial strain in a culture medium, which is amethod in which at least one chromogen that is a substrate for theenzyme of the bacterial strain and at least one compound of a highconcentration, selected from carbohydrates, are added to a culturemedium, and after the hydrolysis of the chromogen, an induced colordifferent from the basic color of the chromophore is obtained. Thismethod is intended to be one in which, for an enzyme that is produced byeach of the bacteria including Candida albicans and Candida tropicalis,two chromogens, which are substrates of the enzyme, are added to aculture medium, and after the hydrolysis of the chromogens, colorsdifferent from the basic color of the chromophore are obtained to carryout distinguishment between the two strains.

CITATION LIST Patent Documents

[Patent Document 1] Japanese Unexamined Patent Application, FirstPublication No. H06-78793

[Patent Document 2] Published Japanese Translation No. H09-500790 of thePCT international Publication

SUMMARY

Among the Candida bacteria, Candida auris, discovered and named by theinventors of the present invention have acquired multiple drugresistance and exhibited a high mortality rate, which has posed a globalthreat. There is a particular need for a means for conveniently andquickly examining whether or not this bacterial species, among theCandida bacteria, is present in a specific environment (for example, acertain closed space such as a hospital room).

For identifying Candida auris with respect to other bacteria, such asother bacterial species of Candida of the Candida bacteria, theidentification can be carried out, for example, by analyzing the genesof the bacteria. However, this analysis requires equipment which may beinconvenient, and requires time and cost as well.

In the detection technique using the ability to degrade an enzymesubstrate such as Patent Document 2 described above, it is possible tocarry out detection with relatively convenient equipment and operationin a short culture time, and in the above technique and a detectionmethod applying the above technique, other Candida bacteria such asCandida albicans and Candida tropicalis can be detected. However,according to the above technique, a method of distinguishing Candidaauris from other Candida bacteria has not been found, and thus Candidaauris cannot be detected by this technique.

For this reason, a technique for conveniently and quickly examining theexistence of Candida auris has not yet been found.

The present invention has been made in consideration of the abovecircumstances, and an object thereof is to provide a screening culturemedium that enables a convenient and quick examination for the presenceof Candida auris, and a screening method using the screening culturemedium.

In order to solve the above object, the present invention has thefollowing aspects.

According to an embodiment, there is provided a screening culture mediumcontaining an enzyme substrate, in which the screening culture medium isused for screening Candida auris based on an ability to degrade theenzyme substrate, the enzyme substrate including raffinose and xylose.

According to at least one embodiment, any of the above screening culturemediums, in which the Candida auris is an East Asian strain of Candidaauris, and the enzyme substrate further includes N-acetylglucosamine

According to at least one embodiment, any of the above screening culturemediums, in which the Candida auris is an East Asian strain of Candidaauris, and the enzyme substrate further includes potassium gluconate.

According to at least one embodiment, any of the above screening culturemediums, in which the enzyme substrate further includes glycerol.

According to at least one embodiment, any of the above screening culturemediums, in which the enzyme substrate further includes D-glucosaminehydrochloride.

According to at least one embodiment, any of the above screening culturemediums, in which each of the enzyme substrates is labeled such that adifferent color is developed in a case of being degraded.

According to another embodiment, there is provided a screening methodincluding screening a microorganism based on an ability to degrade anenzyme substrate, in which in a case where the enzyme substrate includesraffinose and the microorganism has a positive ability to degraderaffinose, and in a case where the enzyme substrate includes xylose andthe microorganism has a negative ability to degrade xylose, themicroorganism is determined to be Candida auris.

According to at least one embodiment, any of the above screeningmethods, in which in a case where the enzyme substrate further includesN-acetylglucosamine, and the microorganism has a negative ability todegrade N-acetylglucosamine, the microorganism is determined to be anEast Asian strain of Candida auris.

According to at least one embodiment, any of the above screeningmethods, in which in a case where the enzyme substrate further includespotassium gluconate, and the microorganism has a negative ability todegrade potassium gluconate, the microorganism is determined to be anEast Asian strain of Candida auris.

According to at least one embodiment, any of the above screeningmethods, in which in a case where the enzyme substrate further includesglycerol, and the microorganism has an ability to degrade glycerol bymore than 0% and 20% or less, which is negative, the microorganism isdetermined to be an East Asian strain of Candida auris.

According to at least one embodiment, any of the above screeningmethods, in which in a case where the enzyme substrate further includesglycerol, and the microorganism has a positive ability to degradeglycerol, the microorganism is determined to be a South American,African, or South Asian strain of Candida auris.

According to at least one embodiment, any of the above screeningmethods, in which in a case where the enzyme substrate is D-glucosaminehydrochloride, and the microorganism has a negative ability to degradeD-glucosamine hydrochloride, the microorganism is determined to be anEast Asian strain of Candida auris.

According to at least one embodiment, any of the above screeningmethods, in which in a case where the enzyme substrate is D-glucosaminehydrochloride, and the microorganism has an ability to degradeD-glucosamine hydrochloride by 80% or more and less than 100%, which ispositive, the microorganism is determined to be a South American strainof Candida auris.

According to at least one embodiment, any of the above screeningmethods, in which in a case where the enzyme substrate is D-glucosaminehydrochloride, and the microorganism has a positive ability to degradeD-glucosamine hydrochloride, the microorganism is determined to be anAfrican or South Asian strain of Candida auris.

According to at least one embodiment, any of the above screeningmethods, further including a step of inoculating a sample in a screeningculture medium containing the enzyme substrate; a step of culturing amicroorganism contained in the sample in the screening culture medium;and a step of detecting the microorganism in the screening culturemedium.

According to at least one embodiment, any of the above screeningmethods, in which a screening culture medium is used, where thescreening culture medium enables detection of an ability of themicroorganism to degrade the enzyme substrate, by allowing each enzymesubstrate to develop a different color.

According to at least one embodiment, any of the above screeningmethods, in which in the step of culturing the microorganism, theculture is carried out at 35° C. or higher.

Advantageous Effects of Invention

According to the present invention, it is possible to obtain a screeningculture medium that enables a convenient and quick examination for thepresence of Candida auris, and a screening method using the screeningculture medium.

DETAILED DESCRIPTION

Hereinafter, a screening culture medium and a screening method accordingto the present invention will be described with reference toembodiments. However, the present invention is not limited to thefollowing embodiments.

Screening Culture Medium Culture Medium Containing RAF and XYL

The screening culture medium according to the present embodimentcontains an enzyme substrate, where the screening culture medium is usedfor screening Candida auris based on the ability to degrade the enzymesubstrate, the enzyme substrate including raffinose and xylose.

Screening broadly refers to an operation of sorting out a microorganism.It broadly refers to an operation of sorting out a sample to be screenedcontaining a microorganism, from the stage of the presence or absence ofthe possibility, by the presence or absence of a certain microorganism.The screening also refers to a step of obtaining more information fordetermining the possibility that a sample contains a certainmicroorganism (for example, determining the possibility from the resultsof a plurality of screenings).

The screening operation may be carried out one time or a plurality oftimes. Alternatively, the same screening operation may be carried out aplurality of times, and this series of operations may be collectivelyreferred to as screening.

The screening culture medium according to the present embodiment is usedfor screening Candida auris.

There are various strains of Candida auris, which are classified intoclades such as a South American clade, an African clade, an East Asian(Japanese and Korean) clade, and a South Asian (Indian) clade, dependingon the origin thereof. According to the screening culture mediumaccording to the present embodiment, any strain belonging to anycladecan be screened as long as it is Candida auris.

According to the screening culture medium according to the presentembodiment, it is possible to carry out screening for the presence orabsence of a certain bacterium or the bacterium classification (theclassification on whether or not a microorganism is a certainbacterium), specifically, depending on whether or not a certainmicroorganism has an enzyme that degrades a specific compound as asubstrate, that is, whether or not it has an ability to degrade aspecific enzyme substrate. The degradation ability is also referred toas assimilation in a case where a microorganism uses an enzyme substrateas a nutrient. Examples of the enzyme substrate that is degraded by abacterium include various carbohydrates (sugars) that are used in thepresent embodiment.

As such a screening culture medium, it is possible to use, for example,a known enzyme substrate culture medium. The enzyme substrate culturemedium is a culture medium in which an enzyme substrate labeled with achromogen (a color-developing substance) is contained, where the enzymesubstrate is a substrate specifically metabolized (degraded andassimilated) by a target microorganism (a bacterial species). In a casewhere an enzyme substrate is incorporated and metabolized by a targetbacterial species, a chromogen dissociates and the condensed chromogendevelops a color (is colored). As a result, it is possible to screen abacterial species having an ability to degrade the enzyme substrate.

As the chromogen with which an enzyme substrate is labeled, it ispossible to use, for example, a compound that releases by hydrolysis twodifferent chromophores that are capable of causing a coupling reaction.As such a compound, it is possible to use, for example, an indoxylderivative. In a case where a culture medium contains a plurality ofenzyme substrates, it is preferable to use a chromogen that exhibits adifferent color for each enzyme substrate, for example, a chromogen of adifferent compound.

The screening culture medium according to the present embodimentcontains raffinose (RAF) and xylose (XYL) among the enzyme substrates.

Candida auris exhibits a positive ability to degrade raffinose and anegative ability to degrade xylose. Specifically, all of the 44 strainsof Candida auris, including each of the above clades, exhibit positivityto raffinose (positive rate: 100%) and negativity to xylose (positiverate: 0%). As a result, in a case where a microorganism cultured in thescreening culture medium of the present embodiment exhibits a positiveability to degrade raffinose and a negative ability to degrade xylose,there is a high possibility that such a microorganism is Candida auris.In addition, the combination of the positive ability to degraderaffinose and the negative ability to degrade xylose is not found inmajor pathogenic bacteria other than Candida auris. As a result, theculture medium of the present embodiment can be used for screening forCandida auris.

In a case where the screening culture medium is an enzyme substrateculture medium, it is preferable that the culture medium containraffinose and xylose each labeled with a chromogen that exhibits adifferent color at the time of degradation.

According to the above configuration, it is possible to carry outscreening such that, among the colonies of microorganisms cultured onthe solid culture medium, a colony that exhibits a color obtained in acase where raffinose is degraded and does not exhibit a color obtainedin a case where xylose is degraded is Candida auris, and a colony thatexhibits only the color obtained in a case where xylose is degraded orboth the colors obtained in a case where raffinose and xylose aredegraded is not Candida auris.

Culture Medium Containing Other Substrates

The enzyme substrate contained in the screening culture medium of thepresent embodiment may further contain another enzyme substrate inaddition to raffinose and xylose. As the enzyme substrate, enzymesubstrates that are used in the related art for identifying a bacterialspecies can be appropriately selected. As a preferred example thereof,the screening culture medium may contain N-acetylglucosamine (NAG),potassium gluconate (GNT), glycerol (GLY), or D-glucosaminehydrochloride (GLN). As will be described later regarding the screeningmethod, in a case where the screening culture medium contains theseenzyme substrates in addition to raffinose and xylose, it is possible toobtain the information on which clade of strain a microorganism belongsto, in addition to the information on whether it is Candida auris.

In a case where the screening culture medium is the enzyme substrateculture medium described above and contains another enzyme substrateother than raffinose and xylose, the other enzyme substrate ispreferably labeled to develop a color different from those of raffinoseand xylose. According to this above configuration, it is possible toobtain the information on which clade of strain a cultured microorganismbelongs to, concurrently with the information on whether or not it isCandida auris, based on the coloration that exhibits an ability todegrade each of raffinose, xylose, and another enzyme substrate (such asN-acetylglucosamine).

Other Configurations

In a case where the screening culture medium of the present embodimentis an enzyme substrate culture medium, other configurations thereof maybe those that are used for the existing culture medium. For example,components can be appropriately selected from the compositions of knownenzyme substrate culture mediums, for example, a color-developingmedium. The enzyme substrate culture medium may contain nutrients suchas peptone, yeast extract, sugar, minerals, and vitamins As thenutrient, peptone is suitably used. The enzyme substrate culture mediummay contain a thickening agent, a pH-adjusting agent, or the like.Further, it may contain an additive for improving the color developmentof the enzyme substrate, for example, a redox reagent.

The enzyme substrate culture medium may contain an antibacterial agentfor suppressing the growth of microorganisms other than the targetmicroorganism for screening. Examples of the antibacterial agent includean aminoglycoside-based antibiotic, a broad-spectrum antibiotic such aschloramphenicol or tetracycline, and cephalosporins or penicillin.Chloramphenicol is suitably used as an antibiotic for screening Candidaauris.

The enzyme substrate culture medium may take an existing form asappropriate, such as liquid, semi-fluid, or solid; however, for example,in a case where a solid culture medium, particularly a flat plate solidculture medium (having a plate shape) is used, culture and detection areeasy. A solidifying agent that is used in the related art, such as agar,carrageenan, or locust bean gum can be used to form a solid culturemedium, and in the present embodiment, agar is used.

It is to be noted that a case where the screening culture mediumcontains a plurality of enzyme substrates may be a case of a combinationof a plurality of culture media containing one or more of the pluralityof enzyme substrates, in addition to a case where one culture mediumcontains all the enzyme substrates. For example, in a case where onekind of microorganism has been isolated in advance and a sample containsonly that microorganism, and in a case where the information on whetherthe microorganism is Candida auris or the information on other clades ofthe microorganism is to be obtained, the present embodiment alsoincludes an aspect in which the sample is cultured in different culturemedia each containing a different enzyme substrate and the informationon the ability to degrade each enzyme substrate is obtained.

Screening Method Screening of Candida auris With Culture MediumContaining RAF and XYL

The screening method of the present embodiment is a screening methodincluding screening a microorganism based on an ability to degrade anenzyme substrate, in which in a case where the enzyme substrate includesraffinose and the microorganism has a positive ability to degraderaffinose, and in a case where the enzyme substrate includes xylose andthe microorganism has a negative ability to degrade xylose, themicroorganism is determined to be Candida auris. Specifically, thescreening is carried out by culturing a target microorganism using theabove-described screening culture medium.

According to the screening method of the present embodiment, it ispossible to carry out screening for whether or not a sample containsCandida auris, where the sample contains a plurality of microorganisms,or it is possible to carry out screening for whether or not a certainmicroorganism has a high possibility of being Candida auris.

As the sample containing a plurality of microorganisms, a samplecollected from a specific space in which microorganisms might be presentcan be conceived.

Specifically, the screening method of the present embodiment furtherincludes a step of inoculating a sample in a screening culture mediumcontaining the enzyme substrate; a step of culturing a microorganismcontained in the sample in the screening culture medium; and a step ofdetecting the microorganism in the screening culture medium.

For example, a method of carrying out screening for whether a samplecontaining a plurality of microorganisms contains Candida auris by usingthe above-described enzyme substrate culture medium will be described.

First, a step of inoculating a sample in a screening culture mediumcontaining the enzyme substrate is carried out. Inoculation may beappropriately carried out according to the cell culture method in therelated art.

Then, a step of culturing the microorganisms contained in the sample iscarried out. Specifically, by culturing the microorganisms in the enzymesubstrate culture medium inoculated with the sample, colonies of themicroorganisms are formed on the enzyme substrate culture medium.

The conditions for carrying out culture in the enzyme substrate culturemedium can be appropriately selected from the conditions for culturingmicroorganisms, known in the related art. In the present embodiment, itis preferable to carry out the culture at 35° C. or higher. In a case ofcarrying out the culture at 35° C. or higher, it is possible to culturepathogenic bacteria that grow under the conditions close to the humanbody temperature, thereby eliminating microorganisms that do not grow atlower temperatures. It is also preferable to carry out the culture at atemperature higher than 40° C. and lower than 42° C. Since Candida auriscan grow even at 41° C., it is possible to eliminate othermicroorganisms that grow at a temperature up to 40° C. by carrying outculture under these temperature conditions.

The culture time can be appropriately selected from the conditions forculturing microorganisms, known in the related art; however, the cultureis carried out for, for example, 24 hours or more and preferably 48hours or more. In a case of carrying out culture for the above time, itis possible to form colonies sufficiently to the extent that colorationcan be checked.

Then, a step of detecting the microorganisms in the screening culturemedium is carried out. In the present embodiment, in a case of checkingthe coloration of the colonies of the microorganisms formed on theenzyme substrate culture medium, it is possible to carry out screeningfor whether the microorganism of the colony is Candida auris. Regardingthe checking of the coloration, it is sufficient to visually checkwhether or not the color of the color-developing body with which theenzyme substrate is labeled is developed for each colony of themicroorganism.

In the above-described enzyme substrate culture medium, a screeningculture medium is used, where the screening culture medium enables thedetection of the ability of the microorganism to degrade the enzymesubstrate, by allowing each enzyme substrate to develop a differentcolor. That is, in the enzyme substrate culture medium of the presentembodiment, the enzyme substrates raffinose and xylose are each labeledto develop a different color in a case of being degraded. As describedabove, Candida auris exhibits an ability to degrade raffinose but doesnot exhibit an ability to degrade xylose. As a result, it can bedetermined that among the colonies of microorganisms cultured on theabove culture medium, a colony that exhibits a color that is developedin a case where raffinose is degraded and does not exhibit a color thatis not exhibited in a case where xylose is degraded is Candida auris. Ina case where a sample containing a plurality of microorganisms has beeninoculated in an enzyme substrate culture medium, and in a case where acolony that shows this coloration is included in the cultured colonies,it can be determined that the sample contains Candida auris.

Screening of Bacterial Clade With Culture Medium Containing OtherSubstrates)

In a case where the screening culture medium of the present embodimentfurther contains glucosamine (NAG), among the Candida auris strains, astrain of the East Asian clade exhibits negativity toN-acetylglucosamine, and strains of the South American, African, andSouth Asian clades exhibit positivity thereto. As a result, in a case ofusing this culture medium, it is possible to screen a strain of the EastAsian clade of Candida auris.

Regarding the clades of Candida auris, it is known that other cladesamong these clades are more pathogenic than the East Asian clade.Regarding the East Asian clade, it is conceived that a fatal infectiondoes not necessarily occur since the infection is limited to localinfection that is basically limited to the inner ear and the outer ear.

That is, in a case of carrying out screening for whether or not acertain microorganism is Candida auris and also on whether or not itbelongs to the East Asian clade, it is possible to obtain theinformation on the risk of the microorganism, or the sample containingthe microorganism, or the environment in which the sample has beencollected.

Further, in a case where the screening culture medium of the presentembodiment contains potassium gluconate (GNT), among the Candida aurisstrains, a strain of the East Asian clade exhibits negativity toN-acetylglucosamine, and strains of the South American, African, andSouth Asian clades exhibit positivity thereto. As a result, in a case ofusing this culture medium, it is possible to obtain the information forscreening a strain of the East Asian clade of Candida auris.

In addition, in a case where the screening culture medium of the presentembodiment contains glycerol (GLY), among the Candida auris strains,only some of strains (roughly, more than 0% and less than 20%) of theEast Asian clade exhibit positivity to glycerol, and strains of theSouth American, African, and South Asian clades exhibit positivitythereto. As a result, in a case of using this culture medium, it ispossible to obtain the information for screening a strain of the EastAsian clade of Candida auris and other strains.

In addition, in a case where the screening culture medium of the presentembodiment contains D-glucosamine hydrochloride (GLN), among the Candidaauris strains, a strain of the East Asian clade exhibits negativity toD-glucosamine hydrochloride, strains of the African and South Americanclades exhibit positivity thereto, and a large number of strains(roughly, more than 80% and less than 100%) of the South Asian cladesexhibit positivity thereto. As a result, in a case of using this culturemedium, it is possible to obtain the information for screening a strainof the East Asian clade of Candida auris and other strains.

In a case where the screening culture medium is the enzyme substrateculture medium described above and contains another enzyme substrateother than raffinose and xylose, and furthermore, the other enzymesubstrate is labeled to develop a color different from those ofraffinose and xylose, it is possible to carry out screening for whetheror not a cultured microorganism is Candida auris and concurrently forwhether or not it is the East Asian clade, based on the coloration thatexhibits an ability to degrade each of raffinose, xylose, and anotherenzyme substrate (such as N-acetylglucosamine)

The screening based on such coloration can also be carried out byappropriately combining two or more substrates each providing differentcoloration. For example, in a case where a screening target is positiveto two or more substrates, two or more types of coloration areexhibited, and a microorganism colony exhibits a color resulting fromthe mixed coloration, whereby it is possible to obtain the informationon which substrate reacts with the screening target, that is, whichstrain of cladeamong Candida auris strains is included, based on thecolor tone and intensity of the colony.

Effect of Present Embodiment

The screening culture medium and screening method according to thepresent embodiment enables a convenient and quick examination for thepresence of Candida auris. Specifically, for a sample containing aplurality of microorganisms, for example, a sample collected from aspecific space, it is possible to carry out screening for the presenceof Candida auris with a convenient operation of just inoculating andculturing the sample in a flat plate solid culture medium and in a shorttime of about 24 to 48 hours. For inoculation and culturing, it sufficesthat such equipment that enables culture in a flat plate solid culturemedium is provided. Furthermore, the screening of microorganisms iscarried out by only visual observation of the coloration, and thusspecial equipment or instrument is not required. Screening with a flatplate solid culture medium can be carried out at a very low cost ascompared with gene analysis and the like.

Application Example of Present Embodiment

As the sample to be screened in the present embodiment, for example, asample collected from a specific indoor or outdoor space can beconceived. Examples of the specific space include places where Candidaauris should be detected, and they include rooms in a hospital,particularly operating rooms or hospital rooms. In addition, those fromvarious facilities or welfare facilities with elderly people and sickpeople can be used. In a case where the detection is carried out at anairport or a customhouse, it can be used for so-called border controlmeasures.

The screening culture medium and the method of the present embodimentcan be used to obtain information on the presence/absence of Candidaauris or any clade of bacteria by combining a screening operation aplurality of times.

In addition, the screening culture medium and the method of the presentembodiment can be used in combination with other means. For example, thescreening culture medium and the method of the present embodiment can beused as a pre-stage screening for narrowing down the target to besubjected to further detailed gene analysis. Since the screening methodof the present embodiment can be carried out conveniently and quickly,it is particularly effective in a case where the number or the like ofsamples to be screened (specific spaces where the presence is examined),clades, or the like is large.

Other Embodiments

The screening method of the present embodiment can also be used to carryout screening for one kind of microorganism on whether the bacterium isCandida auris. In that case, it is also possible to carry out screeningusing an enzyme substrate culture medium containing the above-describedraffinose and xylose or containing another enzyme substrate. Inaddition, the present embodiment also includes an aspect in which theculture is carried out in different culture media each containing adifferent enzyme substrate and the information on the ability to degradeeach enzyme substrate is obtained.

For the enzyme substrate culture medium, a plate-shaped culture mediumis easy to use for culturing microorganisms; however, another form maybe used. For example, a culture medium may be formed into a thin filmshape. Since it is not difficult to simply install a film-shaped culturemedium in various places and culture as is for carrying out screening,it is possible to install the film-shaped culture medium in theabove-described specific space where Candida auris should be detected.

EXAMPLES

Hereinafter, Examples will be described. It is to be noted that thepresent invention is not limited to the following Examples.

Verification of Enzyme Substrate Specific to Candida auris

The ability to degrade each enzyme substrate was examined for aplurality of bacterial strains including Candida auris. As Candidaauris, 44 strains shown in Table 1 (as the bacterial species, 10 strainsof the South American clade, 10 strains of the African clade, 10 strainsof the East Asian clade, and 14 strains of the South Asian clade) wereused. Other bacterial strains were selected from among therepresentative pathogenic yeast bacterial strains in Japan. Thebacterial strains used as strains other than Candida auris are shown inTable 2.

As a kit that can identify the degradation ability as a positive rate, aplate of culture identification/fungus kit ID 32C API (Sysmex BioMérieuxCo., Ltd.) was used for various enzyme substrates including raffinose(RAF), xylose (XYL), N-acetylglucosamine (NAG), potassium gluconate(GNT), glycerol (GLY), and D-glucosamine hydrochloride (GLN) among theenzyme substrates.

Bacterial solutions of the strains shown in Tables 1 and 2 were preparedwith a suspension medium (a liquid culture medium) according to theprotocol and taken into a positive rate detection cup of each enzymesubstrate on a plate of ID 32C API and cultured for 48 hours. Afterculturing, wells that were transparent similar to the control werejudged as negative (−), and wells that were opaque as compared with thecontrol were judged as positive (+) by visual determination, and thenthe obtained positive data were analyzed using the database software 4.0attached to ID 32C API.

TABLE 1 Test Bacterial example strain Clade RAF XYL NAG GNT GLY GLN 1 C.auris #16 South American + − + + + + 2 C. auris #17 South American +− + + + + 3 C. auris #18 South American + − + + + + 4 C. auris #19 SouthAmerican + − + + + + 5 C. auris #20 South American + − + + + + 6 C.auris #21 South American + − + + + + 7 C. auris #22 South American +− + + + + 8 C. auris #23 South American + − + + + + 9 C. auris #24 SouthAmerican + − + + + + 10 C. auris #25 South American + − + + + + SouthAmerican positive rate 100 0 100 100 100 100 11 C. auris #26 African +− + + + + 12 C. auris #27 African + − + + + + 13 C. auris #28 African +− + + + + 14 C. auris #29 African + − + + + + 15 C. auris #30 African +− + + + + 16 C. auris #31 African + − + + + + 17 C. auris #32 African +− + + + − 18 C. auris #33 African + − + + + + 19 C. auris #34 African +− + + + + 20 C. auris #35 African + − + + + + African positive rate 1000 100 100 100 90 21 C. auris #43 East Asian + − − − + − 22 C. auris #44East Asian + − − − − − 23 C. auris 3540 East Asian + − − − − − 24 C.auris 3541 East Asian + − − − − − 25 C. auris 3435 East Asian + − − − −− 26 C. auris 3436 East Asian + − − − − − 27 C. auris 3449 East Asian +− − − − − 28 C. auris 643  East Asian + − − − − − 29 C. auris 3212 EastAsian + − − − − − 30 C. auris 3213 East Asian + − − − − − East Asianpositive rate 100 0 0 0 10 0 31 C. auris #52 South Asian + − + + + + 32C. auris #53 South Asian + − + + + + 33 C. auris 3214 South Asian +− + + + + 34 C. auris 3215 South Asian + − + + + + 35 C. auris 3216South Asian + − + + + + 36 C. auris 3217 South Asian + − + + + + 37 C.auris 3218 South Asian + − + + + + 38 C. auris 3219 South Asian +− + + + + 39 C. auris 3220 South Asian + − + + + + 40 C. auris 3221South Asian + − + + + + 41 C. auris 3222 South Asian + − + + + + 42 C.auris 3223 South Asian + − + + + + 43 C. auris 3224 South Asian +− + + + + 44 C. auris 3225 South Asian + − + + + + South Asian positiverate 100 0 100 100 100 100 Total positive rate 100 0 77 77 80 75

RAF- XYL- Probability of RAF positive positive positivity and XYL rate(%) rate (%) negativity (%) Candida albicans 1 1 98  0.00% Candidaalbicans 2 0 67  0.00% Candida dubliniensis 1 3  1.00% Candida famata 9375  23.30% Candida glabrata 0 1  0.00% Candida globosa 60 0  60.00%Candida guilliermondii 100 99  1.00% Candida intermedia 85 99  0.90%Candida kefyr 99 83  16.80% Candida krusei 0 1  0.00% Candida lusitaniae7 94  0.40% Candida parapsilosis 1 96  0.00% Candida sake 2 74  0.50%Candida tropicalis 9 99  0.10% Cryptococcus neoformans 81 81  15.40%Rhodotorula minuta 0 86  0.00% Candida auris 100 0 100.00%

As shown in Table 1, all of the 44 strains of 4 clades of Candida aurishad a positive ability to degrade RAF and a negative ability to degradeXYL.

Table 2 shows that all of the 44 strains of Candida auris (the bottomrow of the table) had a positive ability (100%) to degrade RAF and anegative ability (0%) to degrade XYL. On the other hand, as shown in theother rows of Table 2, in the pathogenic bacterial strains other thanCandida auris, the combination of a positive ability of 100% to degradeRAF and a negative ability (positive ability: 0%) to degrade XYL was notfound.

That is, among the major pathogenic bacterial strains, only Candidaauris had a positive ability of 100% to degrade RAF and a negativeability of 100% to degrade XYL. It was seen that it is possible to carryout screening for the presence or absence of Candida auris by detectingthe ability to degrade RAF and the ability to degrade XYL.

As shown in the results shown in Table 1, among the 44 strains ofCandida auris, the East Asian strain was positive to RAF and negative toXYL, as well as 100% negative to NAG.

That is, it was seen that in a case of detecting an ability of a Candidaauris strain to degrade NAG in addition to RAF and XYL, it is possibleto carry out screening for whether it is an East Asian strain or anotherstrain.

Further, as shown in Table 1, the East Asian strain was 0% positive toGNT, whereas the strains other than the East Asian were 100% positivethereto.

The East Asian clade was 10% positive to GLY (that is, only one strainof the 10 East Asian strains was positive). From these results, it isconceived that the strains belonging to the East Asian clade includestrains that are positive in a range of about 0 to 20% with respect toGLY. On the other hand, strains other than the East Asian were 100%positive thereto.

The East Asian clade was 0% positive to GLN, whereas the African andSouth Asian clades were 100% positive thereto, and the South Americanclade was 90% positive thereto. From this, it is conceived that thestrains belonging to the South Asian clade include strains that arepositive in a range of about 80% to 100% with respect to GLY.

That is, a strain having a low positive rate to GLY and a strainnegative to GLN can be distinguished from other strains as the EastAsian clade, and it is conceived that these combinations can be used forscreening judgment of each of the strains.

INDUSTRIAL APPLICABILITY

According to the present invention, it is possible to obtain a screeningculture medium that enables a convenient and quick examination for thepresence of Candida auris, and a screening method using the screeningculture medium.

1. A screening culture medium, comprising: an enzyme substrate, whereinthe screening culture medium is used for screening Candida auris basedon an ability to degrade the enzyme substrate, and wherein the enzymesubstrate includes raffinose and xylose.
 2. The screening culture mediumaccording to claim 1, wherein the Candida auris is an East Asian strainof Candida auris, and the enzyme substrate further includesN-acetylglucosamine.
 3. The screening culture medium according to claim1, wherein the Candida auris is an East Asian strain of Candida auris,and the enzyme substrate further includes potassium gluconate.
 4. Thescreening culture medium according to claim 1, wherein the enzymesubstrate further includes glycerol.
 5. The screening culture mediumaccording to claim 1, wherein the enzyme substrate further includesD-glucosamine hydrochloride.
 6. The screening culture medium accordingto claim 1, wherein each of the enzyme substrates is labeled such that adifferent color is developed in a case of being degraded.
 7. A screeningmethod comprising: screening a microorganism based on an ability todegrade an enzyme substrate, wherein in a case where the enzymesubstrate includes raffinose and the microorganism has a positiveability to degrade raffinose, and in a case where the enzyme substrateincludes xylose and the microorganism has a negative ability to degradexylose, the microorganism is determined to be Candida auris.
 8. Thescreening method according to claim 7, wherein in a case where theenzyme substrate further includes N-acetylglucosamine, and themicroorganism has a negative ability to degrade N-acetylglucosamine, themicroorganism is determined to be an East Asian strain of Candida auris.9. The screening method according to claim 7, wherein in a case wherethe enzyme substrate further includes potassium gluconate, and themicroorganism has a negative ability to degrade potassium gluconate, themicroorganism is determined to be an East Asian strain of Candida auris.10. The screening method according to claim 7, wherein in a case wherethe enzyme substrate further includes glycerol, and the microorganismhas an ability to degrade glycerol by more than 0% and 20% or less,which is negative, the microorganism is determined to be an East Asianstrain of Candida auris.
 11. The screening method according to claim 7,wherein in a case where the enzyme substrate further includes glycerol,and the microorganism has a positive ability to degrade glycerol, themicroorganism is determined to be a South American, African, or SouthAsian strain of Candida auris.
 12. The screening method according toclaim 7, wherein in a case where the enzyme substrate is D-glucosaminehydrochloride, and the microorganism has a negative ability to degradeD-glucosamine hydrochloride, the microorganism is determined to be anEast Asian strain of Candida auris.
 13. The screening method accordingto claim 7, wherein in a case where the enzyme substrate isD-glucosamine hydrochloride, and the microorganism has an ability todegrade D-glucosamine hydrochloride by more than 80% and less than 100%,which is positive, the microorganism is determined to be a SouthAmerican strain of Candida auris.
 14. The screening method according toclaim 7, wherein in a case where the enzyme substrate is D-glucosaminehydrochloride, and the microorganism has a positive ability to degradeD-glucosamine hydrochloride, the microorganism is determined to be anAfrican or South Asian strain of Candida auris.
 15. The screening methodaccording to claim 7, further comprising: a step of inoculating a samplein a screening culture medium containing the enzyme substrate; a step ofculturing a microorganism contained in the sample in the screeningculture medium; and a step of detecting the microorganism in thescreening culture medium.
 16. The screening method according to claim15, wherein a screening culture medium is used, where the screeningculture medium enables detection of the presence of an ability of themicroorganism to degrade the enzyme substrate, by allowing each enzymesubstrate to develop a different color.
 17. The screening methodaccording to claim 15, wherein in the step of culturing themicroorganism, the culture is carried out at 35° C. or higher.